Use of a magnesium isotope for treating hypoxia and a medicament comprising the same

ABSTRACT

The present invention describes the use of the magnetic magnesium isotope  25 Mg 2+  for the manufacture of a medicament for treating hypoxia. Furthermore, the present invention provides a medicament for treating hypoxia comprising the magnetic magnesium isotope  25 Mg 2+  and a porphylleren compound according to formula (I) 
     
       
         
         
             
             
         
       
         
         wherein the residues A 1 , A 2 , A 3 , and A 4  independently are a group containing 1 to 40 carbon atoms and at least one carboxylic group, the residues R, R′ and R″ independently are hydrogen or a group containing 1 to 80 carbon atoms, M is  2  H or an element capable of complexing with pyrrole nitrogen atom and L is a linking group and B is a fullerene residue.

PRIORITY

This application claims the benefit of EP07009882 filed on May 18, 2007,and EP07009881, filed on May 18, 2007, which are incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the use of a ²⁵Mg²⁺ for the manufactureof a medicament for treating hypoxia and the same medicament.

BACKGROUND OF THE INVENTION

All hypoxia-relieving therapies ever tested (ATP, Panangin (Russia), O₂inhalation, Magnesium salts, etc) are not “smart” ones, i.e. theiraction in vivo is uncontrollable by the body itself, i.e. by means ofhomeostasis. In other words, these therapies are only useful when thesigns of hypoxia are already observed in the patient and the drugs canonly be applied after the problem has come up.

In the case of therapies involving the inhalation of oxygen, the factthat the required equipment is not available when needed and thedifficulty and/or danger of dealing with oxygen cylinders should also beconsidered as a significant drawback.

Another drawback of existing hypoxia therapies is the lack ofselectivity regarding the mechanism of treatment, that is, the targettissue for which the treatment specifically works cannot be specified.

Hence, there exists a strong need for a new way of treating hypoxia anda respective medicament, which medicament can supply a compound actingas Active Pharmaceutical Ingredient (API) for treating hypoxia. Thesupply or release should be smart and open the way for effective andeasy-to-use application in the treatment of hypoxia for differenttissues.

SUMMARY OF THE INVENTION

In one embodiment the present invention provides a method of treatinghypoxia in a subject comprising administering magnetic magnesium isotope²⁵Mg²⁺ to the subject.

In another embodiment the present invention provides a medicament fortreating hypoxia comprising a magnetic magnesium isotope ²⁵Mg²⁺ and aporphylleren compound according to formula (I)

wherein the residues A¹, A², A³, and A⁴ independently are a groupcontaining 1 to 40 carbon atoms and at least one carboxylic group, theresidues R, R′ and R″ independently are hydrogen or a group containing 1to 80 carbon atoms, M is 2H or an element capable of complexing withpyrrole nitrogen atom and L is a linking group and B is a fullereneresidue.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the formation of diethyl-3-(trimethylsilyl)phthalate(Product I) by reacting diethyl phthalate with n-butyl lithium andadding trimethylsilyl chloride.

FIG. 2 illustrates conversion of Product I of FIG. 1 intodiethyl-3-formyl-6-(trimethylsilyl)phthalate (Product II) by reacting ofProduct I in the presence of n-buthyl lithium and dimethylformamide.

FIG. 3 illustrates transformation of Product II intodiethyl-6-bromo-3-formylphthalate (Product III) by bromination withN-bromosuccinimide and tetrabutylammonium fluoride.

FIG. 4 illustrates condensing of Product III with pyrrole in2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to produce the porphyrinProduct IV.

FIG. 5 illustrates the transformation of the porphyrin Product IV intoProduct V in the presence of dimethylformamide and POCl₃.

FIG. 6 illustrates the conversion of Product V into Product VI by theone-step combined treatment with 2-prophenyltriphenylphosphoniumbromide, lithium disopropylamide (LDA) and lithium perchlorate.

FIG. 7 illustrates how Product VII is formed by treating Product VI withdimethylaminoacetic acid.

FIG. 8 illustrates the transformation of Product VI into product VII bytreatment with gamma-aminobutyric acid and following addition of NaOHand MgCl₂ the transformation of Product VII into Product VIII.

FIG. 9 illustrates the conversion of Product VIII into Product IX, afullerene porphyrin adduct, and the transformation to the final PMC16nanostructure formed by the treatment Product VIII with C₆₀-fullereneand a Pt catalyst (powder suspension) in pyridine with sonication andreaction of Product IX with FeCl₂, o-dichlorobenzene anddimethylformamide in the presence of heat.

FIG. 10 illustrates a multiexponential two-compartment dynamics of theblood serum [Mg]PMC16 concentration after a single 20 mg/kg i.v.injection in rats.

FIG. 11 illustrates the cell compartment retaining distribution of[⁵⁹Fe]PMC16 caused by a single i.v. administration in rats (30 mg/kg,470-520 Ci/kg).

FIG. 12 illustrates the heart muscle cell compartment retainingdistribution of [⁵⁹Fe]PMC16 caused by a single i.v. administration inrats.

FIG. 13 illustrates the effect of a PMC16-targeted delivery of Mg²⁺ onthe doxorubicin pre-suppressed ATP production in rat myocardium.

FIG. 14 illustrates electron transmitting microphotograms of the ratmyocardiocytic perinuclear areas.

FIG. 15 illustrates synergism of the mitochondrial matrix CK activity,PMC16-related magnesium cations influx and the free protons excessdegree.

FIG. 16 illustrates synergism of the ATP yield, oxygen consumption andthe PMC16-related Mg²⁺ release in the rate heart muscle tissue (30 mg/kgPMC16, i.v., 12 hrs. exposition).

FIG. 17 illustrates a highly selective targeting of PMC16 nanoparticlestowards the rat heart muscle in a course of the long-term administrationof an extra low drug dosage.

FIG. 18 illustrates the rat myocardium tissue respiration affected byDXR and MNA in a course of [²⁵Mg²⁺]PMC16 administration (0.4→0.2→0.1mg/kg i.v.)

DETAILED DESCRIPTION OF THE INVENTION

The use of an API and the respective medicament achieving its smartdelivery should further also open the possibility for treatment ofhypoxia in different parts of the body, even with selective delivery ofthe API to the particular targets. According to preferred embodiments,the heart tissue is the target tissue.

Furthermore, the drug or medicament should have the ability toaccumulate in the desired target tissues for a specific period, and alsothe ability to smartly release after the onset of hypoxia. In otherwords, it can also be used to prevent severe hypoxia and/or prevent orat least postpone the dangerous consequences of hypoxia at least untilfurther measures can be taken.

In particular, it is an object of the present invention to provide amedicament having low toxicity and a good efficiency to treat hypoxiawith tissue selectivity if needed.

These objects are solved by the use of the magnetic magnesium isotope²⁵Mg²⁺ for the manufacture of a medicament for treating hypoxia.

Furthermore, the present invention provides a medicament for treatinghypoxia comprising the magnetic magnesium isotope ²⁵Mg²⁺ and aporphylleren compound according to formula (I)

where the residues A¹, A², A³, and A⁴ independently are a groupcontaining 1 to 40 carbon atoms and at least one carboxylic group, theresidues R, R′ and R″ independently are hydrogen or a group containing 1to 80 carbon atoms, M is 2H or an element capable of complexing withpyrrole nitrogen atom and L is a linking group and B is a fullereneresidue.

A nuclear spin selectivity of the ²⁵Mg²⁺-dependent mitochondrial ATPsynthesis pathways is found to be a background for the corrective andeffective intervention of hypoxia related metabolic disorders, althoughthe present invention is not bound to this theory. The exclusively highcapability of ²⁵Mg²⁺, i.e. the only magnetic magnesium isotope, toactivate merely all kinase-promoted processes of nucleotidesphosphorylation in mitochondria is believed to be a basis for thesuccessful treatment. Surprisingly, the ATP depletion in a cell can becorrected by a targeted delivery of ultra-micro-amounts (pico-grams,i.e. 10⁻¹² g) of ²⁵Mg²⁺ isotope.

According to a preferred aspect of the present invention, the medicamentcan achieve a targeted and selective delivery of the ²⁵Mg²⁺ to anymuscle. Preferably, the medicament corrects the energy metabolismdisorders in myocardiocytes.

Moreover, the medicament may increase the efficiency of the treatment ofthe hypoxia suffering muscles and decreases the required dose of ²⁵Mg²⁺required for the treatment.

According to a special embodiment of the present invention, themedicament preferably comprises a target-selective ion carrier. The ioncarrier may possess a high degree of water solubility and the ability tobe dispersed and retained in lipid layer of biological membranes.

The present invention provides a medicament possessing a selectivedelivery and smart release of the ²⁵Mg²⁺ isotope, e.g., specificstructural, magnetic, and/or chemical properties of the tissue can beused for the selective delivery and smart release. In the sense of thisinvention the term “smart” means that the drug is able to release the²⁵Mg²⁺ isotope only in the case there appears hypoxia in the tissue,i.e. only in the scenario of the disease which leads to acidosis andother symptoms. If not necessary (no hypoxia), there will be no release.

If magnesium isotope ²⁵Mg²⁺ is taken through other methods, a high dosedosage is required to have the necessary dosage at the heart muscle, dueto the homogeneous distribution of the ²⁵Mg²⁺ throughout the body.However, if a magnetic magnesium isotope ²⁵Mg²⁺ is used in complex withthe porphylleren compound of the present invention, due to the selectiveand targeted delivery of the ²⁵Mg²⁺ a low dose of about 0.1 to about0.01 times less than that of the sole ²⁵Mg²⁺ can be used. Additionally,the porphylleren compound primarily releases the ²⁵Mg²⁺ in the case ofacidosis of the cells, while in other cases the isotope is readily usedup by the cells.

According to a preferred embodiment of the invention, the aims of theinvention can be fulfilled through the application of target-selectiveion carriers preferably possessing both high degrees of water solubilityand the ability to be dispersed and retained in lipid layer ofbiological membranes.

According to another preferred embodiment of the invention, the aims ofthe invention can be fulfilled by the application of basic salts of theion carriers with ²⁵Mg²⁺.

According to another preferred embodiment of the invention, the aims ofthe invention can be fulfilled by the application of specially designedand synthesized derivatives of porphyrin-adducted nanocationiteparticles.

According to another preferred embodiment of the invention, thederivatives of porphyrin-adducted nanocationite particles may also beused to deliver and smartly release ²⁵Mg²⁺ or any other cationic speciesof desire to cells.

According to another preferred embodiment of the invention, the ioncarriers possess both high degrees of water solubility (460 mg/ml in thecase of PMC16) and the ability to be dispersed and retained in lipidlayer of biological membranes, due to their chemical structures.

According to another preferred embodiment of the invention the targetselectivity of the ion carrier species can be adjusted regarding thespecific characteristics of the target tissue, the chemical structure ofthe carrier, nature of the metal(s) in the metal porphyry, and themagnetic properties of the carrier.

According to another embodiment of the invention, the choice of metal inthe metal porphyry can affect the target selectivity or the functioningof the drugs.

According to another preferred embodiment of the invention the aims ofthe invention can be fulfilled by most preferably the application of aspecific derivative of the mentioned family namely PMC16(fullerene(C₆₀)-2-(butadiene-1-yl)-tetra(o-γ-aminobutyryl-o-phtalyl)metaloporphyrin,in which the metal in the metal porphyry can be chosen from any desiredmetal(s), in the periodic table and one specific or a mixture of theirisotopes either in the naturally abundant form or in a form enrichedwith one specific isotope, or a combination thereof. The choice of themetal, as said before, can affect the target selectivity or thefunctioning of the drug.

According to another preferred embodiment in case the target tissue isto be that of heart, the most preferred ion carrier is PMC16(fullerene(C₆₀)-2-(butadiene-1-yl)-tetra(o-γ-aminobutyryl-o-phtalyl)metaloporphyrin,in which the metal in the metal porphyry is a magnetic isotope of anydesired metal ion.

According to another preferred embodiment of the present invention, inthe case of the target tissue being be that of heart, the most preferredion carrier is PMC16(fullerene(C₆₀)-2-(butadiene-1-yl)-tetra(o-γ-aminobutyryl-o-phtalyl)ironporphyrin in which the iron ion is most preferably Fe(III).

According to another preferred embodiment of the invention, ²⁵Mg²⁺ maybe delivered to any other desired target in the form of a salt with thePMC16 derivative iron complex, by changing the properties of the drugand/or complex.

According to another preferred embodiment of the invention, thetissue-selectivity of the delivery of the mentioned isotope by PMC16, inthe case of heart can be increased by means of the application of the(fullerene(C₆₀)-2-(butadiene-1-yl)-tetra(o-γ-aminobutyryl-o-phtalyl)metaloporphyrincarriers wherein the metal in the metaloporphyrinis any magnetic elementthat can be selectivity pulled towards the heart muscle.

According to another embodiment of the present invention due to thespecial characteristics of porphyrin derivatives, especially PMC16 inthe form of a complex with ²⁵Mg²⁺, they can accumulate in the targettissue for a certain period of time, and be smartly released in thetarget tissue only after hypoxia attacks.

According to another embodiment of the invention, the release of ²⁵Mg²⁺ion in the target happens through an intelligent mechanism, according towhich the isotope is only released in the case of hypoxia.

According to another embodiment of the invention, ²⁵Mg²⁺ is onlyreleased quantitatively in the target tissue, in the case of the hypoxiaand the consequent acidosis and due to the metabolic decay andtransformation of the drug and due to the properties of the drug.

According to another feature of the invention, the drug may also preventthe energy losses in cells and tissues in case of the prior-to-hypoxiaadministration. So long-term courses of the drug may serve tosimultaneous treatment and prevention of hypoxias in patience with achronic heart failure and related pathologies as well as it might beefficient to prepare people for any anticipated (forthcomings)physiological situation, which may lead to hypoxia (e.g. sports, heavyphysical work, stress).

According to another feature of the present invention, because thebiological membrane functioning requires (ATP, GTP hydrolysis relatedenergy supply), and as long as the cell life span depends on membranefunctioning, the proposed drug may provide an additional “guarantee” fora non-stop (regardless on the oxygen accessibility) energy productionmechanism, in the harmful energy losses in the cell in the case ofhypoxia and hence prolong the life span of a cell.

According to another feature of the present invention, because theexcess of bio-oxidation activity in a cell (including all knownperoxide-forming reactions) harms the cell membranes depriving them ofbeing involved properly in to pathways, then the product proposed mayhelp to minimize the consignment energy losses due to the overactivation of ATP production in ²⁵Mg-²⁺CK reaction.

According to another feature of the present invention, because anincrease in lipid peroxidation rate, may harm not only biologicalmembranes but also the membrane-associated ATP-synthase, and NAD(FAD),dehydrogenates cascade as well, which in turn creates a condition for asharp ATP production, this might be compensated by activation of theCK-dissected ATP synthesis which is supposed to be activated by thepresence of ²⁵Mg²⁺.

According to another feature of the present invention, because bothtough-UV and f-ionizing radiations are known to induce trulychaos-leading devastating impacts towards the eukaryotic cell energymegalopolis the delivery of ²⁵Mg-²⁺ might be also applicable tonormalization of at least the damaged ATP/ADP ratio in irradiated cellsand tissues due to marked capabilities to stimulate the O₂-independentATP synthesis due to over activation of the ADP-substratephosphorylation by ²⁵Mg²⁺ cations.

According to another feature of the invention the method can be used fortreating people suffering hypoxia in the heart muscles as a result ofheart attacks, or for people who run the risk of having heart attacks

Preferably, the medicament provides a basic salt of an ion carrier with²⁵Mg²⁺. E.g., the medicament comprises a compound having a porphyringroup.

Preferred compounds having a porphyrin group are porphylleren compoundaccording to formula (I)

wherein the residues A¹, A², A³, and A⁴ independently are a groupcontaining 1 to 40 carbon atoms and at least one carboxylic group, theresidues R, R′ and R″ independently are hydrogen or a group containing 1to 80 carbon atoms, M is 2H or an element capable of complexing withpyrrole nitrogen atom and L is a linking group and B is a fullereneresidue.

Preferably, the linking group L is a bond or a group containing 1 to 40carbon atoms, more preferably 1 to 10 carbon atoms. According to apreferred aspect of the present invention, the linking group L isderived from a butadiene-yl group.

The residue B is a fullerene residue, preferably a fullerene residuehaving 60 to 80 carbon atoms, more preferably a C₆₀-fullerene. Thesolubility of C₆₀-fullerene is 1.3×10⁻¹¹ mg/ml.

In Formula (I) M is 2H or an element capable of complexing with pyrrolenitrogen atom. These elements are well known in the art and includetransition metals, e.g. iron.

The residues R, R′ and R″ independently are hydrogen or a groupcontaining 1 to 80 carbon atoms, preferably 1 to 10 and more preferably1 to 5 carbon atoms. According to a special embodiment of the presentinvention, the residues R, R′ and R″ are hydrogen.

Preferably, the residues A¹, A², A³, A⁴ independently are a groupaccording to formula (II) and/or (III)

wherein n and m independently are a integer in the range of 0 to 10,A^(a) is nitrogen (N), phosphorus (P), arsenic. (As) or a groupcontaining 1 to 40 carbon atoms, the residues X¹, X² and X³independently are hydrogen, halogen or a group containing 1 to 40 carbonatoms. Preferably, A^(a) is nitrogen (N) or a group containing 1 to 5carbon atoms and the residues X¹, X² and X³ independently are hydrogen,halogen or a group containing 1 to 3 carbon atoms. According to apreferred aspect of the present invention, the residues A¹, A², A³, A⁴independently are a group containing 1 to 10 carbon atoms. Preferably,the residues A¹, A², A³, and A⁴ independently are a group containing atleast one nitrogen atom.

The expression “group having from 1 to 80 carbon atoms” refers toradicals of organic compounds having from 1 to 80 carbon atoms. The sameapplies for the expression “group having from 1 to 40 carbon atoms”.

These groups include, inter alia, alkyl, cycloalkyl, alkoxy,cycloalkoxy, cycloalkylthio, alkenyl, alkanoyl, alkoxycarbonyl groupsand also heteroaliphatic, aromatic and heteroaromatic groups. The groupsmentioned can be branched or unbranched.

According to the invention, aromatic groups are radicals of monocyclicor polycyclic aromatic compounds which preferably have from 6 to 20, inparticular from 6 to 12, carbon atoms. Heteroaromatic groups are arylradicals in which at least one CH group has been replaced by N and/or atleast two adjacent CH groups have been replaced by S, NH or O, withheteroaromatic groups having from 3 to 19 carbon atoms. Aromatic orheteroaromatic groups which are preferred for the purposes of theinvention are derived from benzene, naphthalene, biphenyl, diphenylether, diphenylmethane, diphenyldimethylmethane, bisphenone, diphenylsulfone, thiophene, furan, pyrrole, thiazole, oxazole, imidazole,isothiazole, isoxazole, pyrazole, 1,3,4-oxadiazole,2,5-diphenyl-1,3,4-oxadiazole, 1,3,4-thiadiazole, 1,3,4-triazole,2,5-diphenyl-1,3,4-triazole, 1,2,5-triphenyl-1,3,4-triazole,1,2,4-oxadiazole, 1,2,4-thiadiazole, 1,2,4-triazole, 1,2,3-triazole,1,2,3,4-tetrazole, benzo[b]thiophene, benzo[b]furan, indole,benzo[c]thiophene, benzo[c]furan, isoindole, benzoxazole, benzothiazole,benzimidazole, benzisoxazole, benzisothiazole, benzopyrazole,benzothiadiazole, benzotriazole, dibenzofuran, dibenzothiophene,carbazole, pyridine, bipyridine, pyrazine, pyrazole, pyrimidine,pyridazine, 1,3,5-triazine, 1,2,4-triazine, 1,2,4,5-triazine, tetrazine,quinoline, isoquinoline, quinoxaline, quinazoline, cinnoline,1,8-naphthyridine, 1,5-naphthyridine, 1,6-naphthyridine,1,7-naphthyridine, phthalazine, pyridopyrimidine, purine, pteridine orquinolizine, 4H-quinolizine, diphenyl ether, anthracene, benzopyrrole,benzoxathiadiazole, benzoxadiazole, benzopyridine, benzopyrazine,benzopyrazidine, benzopyrimidine, benzotriazine, indolizine,pyridopyridine, imidazopyrimidine, pyrazinopyrimidine, carbazole,acridine, phenazine, benzoquinoline, phenoxazine, phenothiazine,acridizine, benzopteridine, phenanthroline and phenanthrene, which mayalso be substituted.

Preferred alkyl groups include the methyl, ethyl, propyl, isopropyl,1-butyl, 2-butyl, 2-methylpropyl, tert-butyl group, the pentyl,2-methylbutyl, 1,1-dimethylpropyl, hexyl, heptyl, octyl,1,1,3,3-tetramethylbutyl, nonyl, 1-decyl, 2-decyl, undecyl, dodecyl,pentadecyl and eicosyl group.

Preferred cycloalkyl groups include the cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl group, which may besubstituted by branched or unbranched alkyl groups.

Preferred alkenyl groups include the vinyl, allyl, 2-methyl-2-propenyl,2-butenyl, 2-pentenyl, 2-decenyl and 2-eicosenyl groups.

Preferred alkynyl groups include the ethynyl, propargyl,2-methyl-2-propynyl, 2-butynyl, 2-pentynyl and 2-decynyl groups.

Preferred alkanoyl groups include the formyl, acetyl, propionyl,2-methylpropionyl, butyryl, valeroyl, pivaloyl, hexanoyl, decanoyl anddodecanoyl groups.

Preferred alkoxycarbonyl groups include the methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, tert-butoxycarbonylgroup, hexyloxycarbonyl, 2-methylhexyloxycarbonyl, decyloxycarbonyl ordodecyloxycarbonyl group.

Preferred alkoxy groups include alkoxy groups whose hydrocarbon radicalis one of the abovementioned preferred alkyl groups.

Preferred cycloalkoxy groups include cycloalkoxy groups whosehydrocarbon radical is one of the abovementioned preferred cycloalkylgroups.

Preferred heteroaliphatic groups include the abovementioned preferredcycloalkyl radicals in which at least one carbon unit has been replacedby O, S or an NR^(a) group and R^(a) is hydrogen, an alkyl group havingfrom 1 to 6 carbon atoms, an alkoxy group having from 1 to 6 carbonatoms or an aryl group.

Very particular preference is given according to the invention tobranched or unbranched alkyl or alkoxy groups having from 1 to 20 carbonatoms, preferably from 1 to 12, advantageously from 1 to 16 and inparticular from 1 to 4, carbon atoms and cycloalkyl or cycloalkyloxygroups having from 3 to 20 carbon atoms, preferably 5 or 6 carbon atoms.

Although one or more hydrogen atoms in the abovementioned radicals canbe replaced by halogen atoms, preferably chlorine or fluorine, thiol orhydroxy groups or groups of the general formulae NR^(a)R^(b) andN⁺R^(a)R^(b)R^(c)C, where the radicals R^(a), R^(b) and R^(c) are each,independently of one another, hydrogen, an alkyl group having from 1 to6 carbon atoms, an alkoxy group having from 1 to 6 carbon atoms or anaryl group, un-substituted radicals have been found to be particularlyadvantageous.

The formula (I) discloses the compound as an anion. The anionic chargeof compound can be balanced by any cation, e.g. protons, ammonium ionsand metal ions. Surprisingly, the cation exchange properties (cationiteproperties) of the compound can be used in order to achieve superiorproperties as mentioned above. According to one of the aspects of thepresent invention the compound can be used either in its free form, inthe form of a homogenous or heterogeneous complex of one or some organicor in organic cations of different charges. The term homogenous asexpressed here, refers to the case of presence of identical cations inthe complex form of the compound, while according to our definition theterm heterogeneous is used for the case of presence of different organicand/or inorganic cations of identical or different charges in itsstructure. Preferably, the compound comprises ²⁵Mg²⁺ to balance thecharge of the structure as disclosed in formula (I).

Preferably, the medicament comprises a porphyrin-adducted fullerenehaving improved properties. As used herein, the term “porphyllerencompound” means a porphyrin-adducted fullerene. According to a preferredembodiment of the present invention nano-cationite particles ofporphylleren compounds are provided. Preferably, the compounds of thepresent invention are nano particles having a particle size in the rangeof 0.5 to 20, preferably 1.0 to 5 and more preferably 1.5-2.5 nm.Porphylleren-MC16 (PMC16) and their derivatives are preferred examplesof the compounds provided by the present invention. Preferredporphylleren compounds simultaneously possess both high degrees of watersolubility (460 mg/ml in the case of PMC16) and the ability to bedispersed and retained in lipid layer of biological membranes, due totheir chemical structures.

According to a special aspect of the present invention, the medicamentcomprises fullerene(C₆₀)-2-(butadiene-1-yl)-tetra(o-γ-aminobutyryl-ophtalyl)metaloporphyrin,especially fullerene(C₆₀)-2-(butadiene-1-yl)tetra(o-γ-aminobutyryl-o-phtalyl)ferroporphyrin(PMC16).

Preferably, the medicament comprises a complex of the porphyllerencompound with [²⁵Mg²⁺]. The complex molar ratio of the porphyllerencompound to ²⁵Mg²⁺ can be in the range of 1:1 to 1:4.

The compounds of the present invention preferably have a low toxicity.Preferably, the toxicity of the compound (LD₅₀ is 1000 mg or less, morepreferably the toxicity of the compound LD₅₀ is 2000 mg/kg or less. TheLD₅₀ can be measured by using rats (i.v., known methods).

Preferably, the compound according to the present invention is solublein water. According to a preferred aspect, the solubility is at least300 mg/ml, more preferably at least 450 mg/ml at pH 7.4.

According to a special aspect of the invention, the compound ismembranotropic. Preferably, the claimed compounds may have highcationite capacities (320-370 meqv/mmol (in the case of PMC16)), whichmake them suitable to be used in cationic complexes.

Surprisingly, the selective delivery of ²⁵Mg²⁺ increases the efficiencyof the treatment of the hypoxia suffering muscles and decreases therequired dose of ²⁵Mg²⁺ required for the treatment.

On the other hand, the remaining of the drug in the cell, due to itsstructural and/or chemical properties, and the subsequent smart releaseof the isotope in the case of hypoxia, will give the mechanism otheradvantages like being able to be used as a precaution for probablehypoxia attacks.

The application of target-selective ion carriers preferably possessingboth high degrees of water solubility and the ability to be dispersedand retained in lipid layer of biological membranes in the form of basicsalts of the ion carriers with ²⁵Mg²⁺ is suggested as a preferredmechanism for the delivery of the desired isotope to cells, and thesmart release of the isotope during or (in case the drug is used toprevent the effects of hypoxia) at the onset of hypoxia.

The target selectivity of the ion carrier species is adjusted regardingthe specific characteristics of the target tissue, designing thechemical structure of the carrier, and the nature of the metal(s) in themetal porphyrin. Most preferably the application of a specificderivative of the mentioned family namely PMC16(fullerene(C₆₀)-2-(butadiene-1-yl)tetra(o-γ-aminobutyryl-o-phtalyl)metaloporphyrin, in which the metal in the metal porphyrin can be chosenfrom any desired metal(s), in the periodic table and one specific or amixture of their isotopes either in the naturally abundant form, or inform(s) enriched with one specific isotope, or a combination thereof, isprovided.

In the case of the target tissue to be that of heart, the most preferredion carrier is PMC16(fullerene(C₆₀)-2-(butadiene-1-yl)-tetra(o-γ-aminobutyryl-o-phtalyl)metaloporphyrin, in which the metal in the metal porphyrin is a magneticisotope of any desired metal ion, most preferably or Fe(II) and whichhelps deliver the ²⁵Mg²⁺ complex to any desired target, specially tothat of heart.

PMC16 derivatives, in the form of complex with ²⁵Mg²⁺, can accumulate inthe target tissue or can be designed to do so, for a certain period oftime, decreasing the risks caused by hypoxia and smartly releasing theisotope in the case of a hypoxia.

PMC16, in the form of complex with ²⁵Mg²⁺ can accumulate in the targettissue, especially that of heart, for a certain period, decreasing therisks caused by the unexpected hypoxia attacks.

The release of ²⁵Mg²⁺ ion in the target happens through a smartmechanism, according to which the isotope is released only in the caseof hypoxia and the consequent acidosis of the cell, which leads to thehydrolysis of the drug and hence releases of the isotope.

Preferably, a porphyrinic fullerene based 1.8-2.0 nm magnesium-carryingparticles can be used in order to achieve the objects of the presentinvention, that is: Porphylleren-MC16, in brief PMC16, which is afullerene(C₆₀)-2-(butadiene-1-yl)-tetra(o-γ-aminobutyryl-ophtalyl)ferroporphyrin.

Preferred compounds being useful in order to achieve the objects of thepresent invention are amphiphilic, membranotropic, slowly metabolizingand low toxic nanocationite to release magnesium in response to thetissue acidosis only, i.e. not sooner than the hypoxia induced pH acidicshift is in fact occurs in a suffering cell. This “smart behavior” ofthe particles proposed allowed to use them as an appropriate tool inprevention/correction of various tissue hypoxia-related ATP-depletingsyndromes that harms myocardium in Mammals.

In order to improve the targeted drug delivery purposes, some steps canbe taken to apply a binding protein receptor for the compound beinguseful to deliver the ²⁵Mg²⁺, e.g. a PMC16-binding protein receptor forthe drug targeting; a receptor mentioned is a specific compound of theheart muscle mitochondria membranes (presumably homologous to themyoglolin B-chain), which provides them an ability to retain the drugmolecule for not less than a week after a single i.v. Injection.

The drug may also prevent the energy losses in cells and tissues in caseof the prior-to-hypoxia administration. So long-term courses of the drugmay serve to simultaneous treatment and prevention of hypoxias inpatience with a chronic heart failure and related pathologies as well asit might be efficient to prepare people for any anticipated(forthcomings) physiological situation, which may lead to hypoxia (e.g.sports, heavy physical work, stress).

Because the biological membrane functioning requires (ATP, GTPhydrolysis related energy supply), and as long as the cell life spandepends on membrane functioning, the proposed drug will provide anadditional “guarantee” for a non-stop (regardless on the oxygenaccessibility) energy production mechanism. In the harmful energy lossesin the cell in the case of hypoxia and hence prolong the life span of acell.

Because the excess of bio-oxidation activity in a cell (including allknown peroxide-forming reactions) harms the cells membranes, deprivingthem of being involved properly into pathways, the product may helpminimize the consignment energy losses due to the over activation of ATPproduction in ²⁵Mg-²⁺CK reaction.

Increase in lipid peroxidation rate, may harm not only biologicalmembranes the membrane-associated ATP-synthase and NAD(FAD),Dehydrogenates cascade as well, which in turn create a condition for asharp ATP production. This might be compensated by activation of theCK-dissected ATP synthesis, which is supposed to be activated by THEpresence of ²⁵Mg²⁺.

Both tough-UV and f-ionizing radiations are known to induce a trulychaos-leading devastating impact towards the eukaryotic cell energymegalopolis the delivery of ²⁵Mg-²⁺ might be also applicable tonormalization of at least the damaged ATP/ADP ratio in irradiated cellsand tissues due to marked capabilities to stimulate the O₂-independentATP synthesis due to over activation of the ADP-substratephosphorylation by ²⁵Mg²⁺ cations.

As an application of the present drug it can be used for treating peoplesuffering hypoxia in the heart muscles as a result of heart attacks, orfor people who run the risk of having heart attacks if the drug can beselectively delivered to the heart tissue.

The synthesis of the preferred porphylleren compound can be carried outaccording to any suitable procedures. Preferably, the method includes alight induced reaction of the reactants under other desired andoptimized reaction conditions and parameters mentioned below.

According to another embodiment of the present invention, any propersolvent or reaction medium can be used in the overall synthesisprocedure. However, due to the high solubility of preferred compoundsaccording to the present invention, e.g. PMC 16, some or all parts ofthe synthesis procedure can be easily and effectively synthesized inaqueous media, which makes the synthesis process environment-friendlyand the product clean from any undesired, harmful solvent of any kind.

The general method designed is as follows. According to a preferredmethod, the molecule can be synthesized in an ultrasonic microwave ovenat a temperature between 50 to 150° C., preferably at 90-120° C. Thereaction is performed under electromagnetic wavelengths between1000-3000 nm, preferably between 1,600-2,200 nm wavelengths withfrequencies in the range of 10-100 KHz, more preferably between 40-60KHz. The preferred reaction time is between 0.5-10 hrs, preferablybetween 1-5 hrs, and most preferably 2.5 hrs. The extensive studies ofthe inventors show that, the presence of catalytic amounts of any propercatalyst, either in the form of a metal powder, metal complex, or anyother organic or inorganic compound may be useful for the properexecution of the synthesis procedure. These studies revealed thatpresence of metallic solid, powder, granular or nano-particles of one ora combination of Pt, Au, Mn, W, Hg, Cu, Pb, or as mentioned any othercatalytic metal, preferably the presence of Pt powder catalystsuspending permanently in pyridine or any other proper organic solventcan be extremely useful for the proceeding of the reaction. Researchperformed by the inventors shows that presence of 5-100μ, preferably10-70μ, and most preferably of 20-40 μmetallic granular Pt powdercatalyst suspending permanently in 40-80% pyridine, (2.0-0.35 mg/cm³)can lead to the best results in terms of yield and purity of the productand reaction parameters.

According to the proposed synthesis method an original modification ofthe classical Prato reaction was employed according to which a simpleDiels-Alder adduct of fullerene C₆₀ or any other proper fullerene familymember, with 2-(butadiene-1-yl)teteraphenylporphyrin or any other properreactant or the mixture of them (depending on the desired derivative)was obtained with a yield of about 97-100%.

The reaction of these diene(s) with C₆₀-fullerene or other desiredfullerene family members was perfectly completed in two hrs, whereascycloadditions with N-phenylmaleimide (or naphtoquinone) usually mayrequire 46-48 hrs.

To generate the above named adduct or any of its proper derivatives, thepyrrolidinefullerene-forming 1,3-dipolar cycloaddition of correspondingazomethynylides was first performed. The latter compounds, in turn, weregenerated by a direct decarboxylation of immonium salts resulting due toa condensation between α-aminoacid (L-prolyne) or its derivatives andthe classical Prato's pyrrolidine-porphyrinic aldehydes.

Being covalently bound to the fullerene family acceptor chromophore, theporphyrin domain played a donor-compound role. The PMC16porphyrin-fullerene diade, or its desired derivative, was completedapart in a way of the porphyrin domain reattachment to the Pratoreaction-generated fullerene derivative,4-(N-methyl-3,4-fullerene-pyrrolydine) phtalenonitryl or any of itsderivatives. This step was carried out at boiling conditions in an inertgas atmosphere, e.g. Ar or other noble gas-aerating, by using puretoluene or any other proper organic or inorganic solvent for 1-6 hrs,preferably 4 hrs.

This step can be followed by a following 2-10 hrs, preferably 6 hrs longincubation of the product with proper concentrations, preferably 1.0-3.0M, most preferably with 2.0 M of any soluble metal salt solution.

The metal solution is chosen according to the needs or desires and canbe chosen from any of the metallic or non-metallic actions, preferably,from soluble solutions of transition or heavy metal salts (withdifferent anions and crystal waters) most preferably that of FeCl₂/1.0 Mdimethylformamide in boiling O-dichlorobenzene or any other propersolvent.

The “loading” of the porphylleren compound with ²⁵Mg²⁺ cations(preparation of PMC16[²⁵Mg²⁺]_(n), where n is the number of ²⁵Mg²⁺ andcan be any number with respect to the structure of the carrier, and ispreferred to be 4 in the case of PMC16), can be done by using solutionscomprising ²⁵Mg²⁺. A typical method, based on the investigations of theinventors, PMC16 “loading” is mentioned in example 1. This is just atypical example and any of its variations are also claimed by theinventors and can be used for the same purpose.

The invention also encompasses use of medicaments for treatment ofhypoxia. The medicament is typically a sterile enclosed container, suchas an ampule, containing a solution of PMC16[²⁵Mg²⁺] in water suitablefor intravenous administration to a human. The solution can be astandard physiological solution (0.9% NaCl₂) adjusted to pH of about7.60 to about 7.80, such as by 0.75 mM NaOH.

The invention is illustrated in more detail below by a preparationexample, without intending to limit the invention to these examples.

EXAMPLE 1 Preparation of PMC16[²⁵Mg²⁺]₄

The compound fullerene(C₆₀)-2-(butadiene-1-yl)-tetra(o-γ-aminobutyrylo-phtalyl)ferroporphyrin(Porphylleren-MC16 or PMC 16) can be pre-pared by the following methodwhich method is exemplified in the scheme as mentioned in FIGS. 1 to 9.

First a diethyl phthalate is to be silicomethylated by thetrimethylsilicochloride in the presence of the lithium butyrate catalystwith a formation of the diethyl-3-(trimethylsilyl)phthalate (product I)in FIG. 1. The latter one is then transforms into thediethyl-3-formyl-6-(trimethylsilyl)phthalate (Product II) by oxidationof the product I in the presence of the same mentioned catalyst and thedimethylazomethanol (see FIG. 2). The product II is then momobromated byN-bromosuccinimide in the presence of the butyrylazofluoride so thediethyl-6-bromo-3-formylphthalate (Product III) formed as a result (seeFIG. 3). Next, the Product III is to be condensated with pyrrol in the2,3-dichloro-5,6-dicyano-1m4-benzoquinone (DDQ) presence which leads toformation of the Product IV, the first obtained porphyrin structure (seeFIG. 4). Then the Product IV transforms into the Product V (see FIG. 5)in the presence of dimethylazomethanol and POCl₃.

Then the Product V transforms into the Product VI by the one-stepcombined treatment with lithium disopropylamide (LDA) and the lithiumperchlorate to form Product VI (see FIG. 6). Dimethylaminoacetic acid isto induce the transformation of the Product VI into the Product VII likeindicated in the scheme (see FIG. 7). Simple addition of NaOH and MgCl₂transforms Product VII into the Product VIII which, in turn, transformesthen into the fullerene porphyrin adduct by the treatment withC60-fullerene, Pt catalyst (powder suspension), here a simple pyridinesonication is involved. Eventually, the final PMC16 nanostructure is tobe formed from the Product IX by incubation (combined simultaneoustreatment) with FeCl₂, butyryldichloride and dimethylazoethanol (seeFIGS. 8 and 9).

PMC16 “loading” with ²⁵Mg²⁺ cations(saturation, PMC16 [Mg²⁺]₄) is doneaccording to a following procedure. Up to 600-800 mg of PMC16 isdissolved in 1.0 mL of mixture of pyridine: CS₂: chloroform or any othersuitable solvents (with a ration of 1:2:7) at the room temperature. Then4 volumes of 2.5M solution of pure ²⁵MgCl₂ (98.6% isotopic purity) in0.2M NAOH is added to the PMC16-containing organic mixture. After 30 minof extensive shaking (at 22-25° C.), the water-soluble phase isseparated by centrifugation at 15000 r.p.m 20 min, 22° C., carefullycollected and then lyophilized (or evaporated). Followingmassspectroscopy and atomic absorption spectroscopy data are used tocontrol the Mg/Fe ratio in the produced PMC16 complex.

The remaining organic phase was repeatedly (2-3 times) subjected to Mg²⁺alkaline water extraction as described above. The organicphase-containing ²⁵MgCl₂ traces are then re-utilized. Using the fastrotor evaporating and a consequent dissolving in 10 mM sodium-phosphate(pH=9.60) buffer containing 15Mm EDTA. The very same way to concentratethe resulted PMC16[Mg²⁺]₄ is applied to the first alkaline-waterextraction run.

EXAMPLE 2 In Vivo Tests on Basic Pharmacokinetics and SomeBioTransformation Patterns of the Drug on Rats

Considering an essential pharmacological potential of PMC16[Mg²⁺]₄, itsbasic pharmacokinetics along with some biotransformation patterns wereestimated by in vivo tests carried out with rats (FIG. 10-13, Table 1).

Besides, a biochemical/pharmacological study on PMC16[Mg²⁺]₄ effects inrat myocardium affected by the drug (Doxorubicin [DXR],1-methylnicotineamide [MNA]) induced hypoxia were conducted to proveboth drug efficiency and safeness with respect to cationite propertiesof the new nano medicament (FIGS. 13,18). Same tests were also performedin inhalation hypoxia model specified below (FIG. 14-16).

The following in vivo experimental hypoxia models were employed:

(1) Medicinal hypoxia syndrome A (1-methylnicotineamide (MNA), 10-20mg/kg i.v. once per 24 hrs); 6858611(2) Medicinal hypoxia syndrome B (DXR 60-80 mg/kg i.v. once per 24 hrs);(3) The oxygen depleted inhalation model (10-20 day long exposition ofrats to the artificial gas mixture to breath with, 12-16% oxygen/84-88%nitrogen, v/v).

Male adult Wistar Albino Glaxo rats (180-220 gms) were used, 3-4 animalsper each experimental point.

As seen from results of all tests conducted (Table 1, FIG. 10-18) theproposed product (PMC16) is indeed efficient for protecting the heartmuscle tissue from the damaging impact promoted by hypoxia of any sort,even when the extra low doses of the drug administrated (FIG. 17).

Particularly, due to the PMC16-specific receptor located in themyocardiocyte mitochondria membranes, a selective targeted delivery ofthe drug might be managed simply by a long term multiple administrationof the drug extra low doses. Being totally safe, this course provides amarked prophylactic and therapeutic effect in medicinal hypoxiccardiotoxicity cases (DXR, MNA)—FIGS. 13, 16,18.

TABLE 1 THE [Mg]PMC16 PHARMACOKINETICS IN RAT Single 20 mg/kg i.v.injection (M ± SEM, n = 6) 24 hrs monitoring presented T_(1/2) = 9.0 hrsC₀ = 62 μg/ml T_(max) = 2.5 hrs C_(max) = 260 ± 83 ng/ml Cl = 32 ± 4ml/min/kg V_(P) = 16.2 ml/kg k = 0.685 V_(C) = 12.4 ml V₁ = 0.08 mlRenal excretion:   28 ± 4.3% Hepatic excretion:   16 ± 4.0%(metabolization) Plasma proteins binding:  1.2 ± 0.3% BLOOD CELLS UPTAKELymphocytes: 28.6 ± 5.5% Erythrocytes:  8.0 ± 3.2% TISSUE SPECIFICACCUMULATION Myocardium:  18.4 ± 3.40% Brain:  0.6 ± 0.02% URINEELIMINATING (258 ± 4.0 μg/ml)  PMC16 METABOLITES Alanyl-depletedderivatives: 56.4 ± 8.7% Deacetylated derivatives: 27.0 ± 6.1%Cyclohexyl-C60: 16.2 ± 3.3% PMC16 urine content: 462 ± 11 μg/ml C60urine content: 2.9 ± 0.1 μg/ml

1. A method of treating hypoxia in a subject comprising administeringmagnetic magnesium isotope ²⁵Mg²⁺ to the subject.
 2. The methodaccording to claim 1 wherein the treatment results in a targeted andselective delivery of the ²⁵Mg²⁺ to a muscle.
 3. The method according toclaim 1 wherein the treatment corrects energy metabolism disorders inmyocardiocytes.
 4. The method according to claim 1 wherein the treatmentis carried out with a medicament that increases the efficiency of thehypoxia suffering muscles and decreases the required dose of ²⁵Mg²⁺required for the treatment.
 5. The method of claim 1, wherein the methodfurther comprises a target-selective ion carrier.
 6. The method of claim5 wherein the ion carrier is water soluble and has ability to bedispersed and retained in lipid layer of biological membranes.
 7. Themethod of claim 1, wherein the method further provides a selectivedelivery of the Mg²⁺ isotope.
 8. The method according to claim 7 whereinspecific structural, magnetic, and/or chemical properties of tissue isused for the selective delivery and smart release.
 9. The method ofclaim 1, wherein the treatment provides a basic salt of an ion carrierwith ²⁵Mg²⁺.
 10. The method of claim 4, wherein the medicament comprisesthe magnesium isotope ²⁵Mg²⁺ in the form of a complex and the complex isable to accumulate in a target tissue, and can be released in the targettissue only after hypoxia attacks.
 11. The method of claim 10, whereinthe release of ²⁵Mg²⁺ ion in the target happens through a mechanism,according to which the isotope is only released in the case of hypoxia.12. The method of claim 10, wherein ²⁵Mg²⁺ is only releasedquantitatively in the target tissue, in the case of the hypoxia and theconsequent acidosis and due to the hydrolysis of the drug.
 13. Themethod of claim 4, wherein the medicament prevents the energy losses incells and tissues in case of the prior-to-hypoxia administration. 14.The method of claim 4, wherein the medicament serves to simultaneoustreatment and prevention of hypoxias in subjects with a chronic heartfailure and related pathologies in long-term courses.
 15. The method ofclaim 4, wherein the medicament provides a non-stop energy productionmechanism by which harmful energy losses in the cell in the case ofhypoxia can be prevented and hence prolongs the life span of a cell. 16.The method of claim 4, wherein the medicament minimizes the consignmentenergy losses due to the over activation of ATP production in ²⁵Mg-²⁺CKreaction.
 17. The method of claim 4, wherein the medicament creates acondition for a sharp ATP production, which might be compensated by theactivation of the CK-dissected ATP synthesis supposed to be activated bythe presence of ²⁵Mg²⁺.
 18. The method of claim 4, wherein the deliveryof ²⁵Mg-²⁺ is applicable to normalization of at least the damagedATP/ADP ratio in irradiated cells and tissues due to marked capabilitiesto stimulate the O2-independent ATP synthesis due to over activation ofthe ADP-substrate phosphorylation by ²⁵Mg²⁺ cations.
 19. The method ofclaim 4, wherein the medicament can be administered to the patients byinhalation, injection or oral application forms.
 20. The method of claim4, wherein the medicament comprises a compound having a porphyrin group.21. A medicament for treating hypoxia comprising a magnetic magnesiumisotope ²⁵Mg²⁺ and a porphylleren compound according to formula (I)

wherein the residues A¹, A² A³, and A⁴ independently are a groupcontaining 1 to 40 carbon atoms and at least one carboxylic group, theresidues R, R′ and R″ independently are hydrogen or a group containing 1to 80 carbon atoms, M is 2H or an element capable of complexing withpyrrole nitrogen atom and L is a linking group and B is a fullereneresidue.
 22. The medicament according to claim 21 wherein the toxicityof the compound LD₅₀ is 2000 mg/kg or less.
 23. The medicament accordingto claim 21 wherein the solubility is at least 450 mg/ml at pH 7.4. 24.The medicament according to claim 21 wherein the compound ismembranotropic.
 25. The medicament according to claim 21 wherein thefullerene residue is a C₆₀ fullerene.
 26. The medicament according toclaim 21 wherein the residues R, R′ and R″ independently are hydrogen ora group containing 1 to 5 carbon atoms.
 27. The medicament according toclaim 21 wherein the residues R, R′ and R″ are hydrogen.
 28. Themedicament according to claim 21 wherein the residues A¹, A², A³, A⁴independently are a group according to formula (II) and/or (III)

wherein n and m independently are a integer in the range of 0 to 10,A^(a) is nitrogen (N), phosphorus (P), arsenic (As) or a groupcontaining 1 to 40 carbon atoms, the residues X¹, X² and X³independently are hydrogen, halogen or a group containing 1 to 40 carbonatoms.
 29. The medicament according to claim 28 wherein A^(a) isnitrogen (N) or a group containing 1 to 5 carbon atoms and the residuesX¹, X² and X³ independently are hydrogen, halogen or a group containing1 to 3 carbon atoms.
 30. The medicament according to claim 21 whereinthe residues A¹, A², A³, A⁴ independently are a group containing 1 to 10carbon atoms.
 31. The medicament according to claim 21 wherein theresidues A¹, A², A³, and A⁴ independently are a group containing atleast one nitrogen atom.
 32. The medicament according to claim 21wherein M is a transition metal.
 33. The medicament according to claim32 wherein M is iron.
 34. The medicament according to claim 21 whereinthe linking group L comprises 1 to 10 carbon atoms.
 35. The medicamentaccording to claim 34 wherein the linking group L is derived from abutadiene-yl group.
 36. The medicament according to claim 21 wherein thecompound is fullerene(C₆₀)-2-(butadiene-1-yl)-tetra(o-γ-aminobutyryl-ophtalyl)ferroporphyrin.37. The medicament according to claim 21 wherein the medicamentcomprises a complex of the porphylleren compound with [²⁵Mg²].
 38. Themedicament according to claim 37 wherein the complex molar ratio of theporphylleren compound to ²⁵Mg²⁺ is in the range of 1:1 to 1:4.